536 Volume 51, Number 4, 1997 APPLIED SPECTROSCOPY0003-7028 / 97 / 5104-0536$2.00 / 0q 1997 Society for Applied Spectroscopy

Structural Characterization of b -Lactoglobulin in SolutionUsing Two-Dimensional FT Mid-Infrared and FTNear-Infrared Correlation Spectroscopy

NELSON L. SEFARA, NOEL P. MAGTOTO, and HUGH H. RICHARDSON*Department of Chemistry, Clippinger Laboratories, Ohio University, Athens, Ohio 45701-2979

Two-dimensional (2D) FT-IR correlation analysis was applied toboth the mid-IR (MIR) and near-IR (NIR) regions to investigatechanges in the secondary structures of b -lactoglobulin in D2O (orH2O) solvent systems consisting of varying concentrations of bro-moethanol. Mid-IR correlation spectra indicate that the amide Ibands corresponding to different structures (i.e., a -helical struc-tures at 1650 cm 2 1, aggregated b -strands at 1620 cm 2 1, and b -sheetat 1636 cm 2 1) exhibit apparently different spectral response towardsvarying concentrations of bromoethanol. We propose that themechanism for the conversion of the b -sheet into a -helix occurs interms of two parallel pathways, i.e., (1) b -sheets ® aggregatedb -strands ® a -helix, and (2) b -sheets ® a -helix. Although the amideB/amide II combination bands give no spectral features relating tothe secondary structure, changes were found in the C± H combina-tion bands that suggest an interaction between the solvent and theprotein.

Index Headings: Protein structure; FT mid-infrared; FT near-infra-red; 2D correlation spectroscopy.

INTRODUCTION

Fourier transform mid-infrared (FT-MIR) studies haveshown that the peak frequencies of the amide bands suchas amide I (predominantly C5 O stretch) and amide II(N± H bending coupled to C± N stretch) can be used todraw conclusions about the secondary structure of pro-teins.1± 4 The frequencies of these vibrational modes havebeen shown to be highly sensitive to the changes in thenature of the hydrogen bond involving C5 O and N± Hgroups of the peptide bond. We carried out experimentsto reveal various transformations in the secondary struc-ture of b -lactoglobulin that accompany changes in theconcentrations of bromoethanol in D2O5 (or H2O). Weused bromoethanol as a perturbing medium because hal-ogenated alcohols have been shown to induce changes inthe secondary structure of proteins.6 In order to obtaindetailed IR spectral information on protein conformation,we have employed 2D correlation data analyses devel-oped for characterizing differences in spectral responsesbetween spectral elements of a set of spectra with certainvariations present among them.7± 9 The spectral bands as-sociated with a -helical and b -sheet structures of b -lac-toglobulin in both the MIR and near-infrared (NIR)regions were correlated, and the results were comparedto previously published data.

EXPERIMENTAL

Samples. Three-times crystallized b -lactoglobulin pur-chased from Sigma Chemical Co. was used without fur-

Received 31 July 1996; accepted 2 January 1997.* Author to whom correspondence should be sent.

ther puri® cation. Fourteen samples were prepared by dis-solving the protein in a solvent system containing bro-moethanol (Aldrich Chemical Co.) and H2O (or D2O,MSD Isotope Co.) to make a ® nal concentration of 20mg/mL. Only solutions with mole fractions (MFs) up to0.300 were analyzed, because of the dif® culty in elimi-nating all the information from the solvent at higherbromoethanol mole fractions.

Instrumentation. FT-MIR spectra were acquired at4-cm2 1 resolution with a Mattson Sirrus 100 spectrome-ter. The protein solutions were prepared for analysis in aPerkin± Elmer demountable cell with CaF2 windows anda 15-m m spacer. On the other hand, NIR spectra werecollected at 2-cm2 1 resolution in a 2-mm-pathlength rect-angular infrared quartz cell with Bio-Rad FTS-60A. Thespectral region from 5000 to 4000 cm2 1 was isolated byusing a multilayer optical interference ® lter (Barr Asso-ciates Co.). A total of 256 scans were collected for eachof the 14 samples for both the MIR and NIR measure-ments. Reference spectra involving only the solvent werecollected under identical conditions.

Data Treatment. All MIR and NIR spectra were base-line corrected with the use of the Grams/386 softwarepackage (Galactic Industries Co.). Synchronous and asyn-chronous MIR and NIR correlation intensities were cal-culated for the 14 spectra with the use of the generalized2D method developed by Noda.7 This method enablesone to study correlations resulting from ¯ uctuations inspectral intensities as a function of any physical variableother than time. Two-dimensional synchronous and asyn-chronous correlation contour maps were plotted with theuse of Matlab (The MathWorks Inc.).

RESULTS AND DISCUSSION

Spectra in the MIR Region. Figure 1 shows the MIRspectra of b -lactoglobulin in bromoethanol/D2O in the1800- to 1500-cm2 1 region (amide I and amide II region).The spectral features of the amide I band are very similarto those previously reported by Jackson and Mantsch.6 Inpure D2O (MF 5 0) the amide I consists of a broad bandcentered around 1636 cm2 1, which is typical of proteinswith a predominantly b -sheet structure.3,10 The amide IIvibration appears around 1575 cm2 1. Increasing the molefraction of bromoethanol from 0.00 to 0.080 leads to thegrowth of two amide I bands around 1620 and 1650 cm2 1

and the rapid decay of the amide II band (1575 cm2 1).We assigned the band at 1620 cm2 1 to aggregatedb -strands and the band around 1650 cm2 1 to the a -helixsecondary structure.5,6 At a much higher bromoethanolmole fraction level (MF 5 0.098± 0.300), the band at

APPLIED SPECTROSCOPY 537

FIG. 1. FT-MIR absorbance spectra in the amide I and II regions forb -lactoglobulin in bromoethanol/D2O. MF 5 mole fraction of bro-moethanol in D2O.

FIG. 2. (A) Synchronous and (B) asynchronous 2D FT-MIR correla-tion contour maps of b -lactoglobulin in bromoethanol/D2O. Shaded ar-eas represent regions of negative correlation.

1650 cm2 1 continued to grow to its maximum peak, whilethe bands at 1636 and 1620 cm2 1 completely disappeared.This result indicates that b -lactoglobulin exists predom-inantly in the a -helix conformation under these condi-tions. The spectral feature observed at 1725 cm2 1 comesfrom the hydrogen-bonded COOH groups.10

Two-Dimensional Correlation in the MIR Region.A synchronous two-dimensional (2D) spectrum is char-acterized by autopeaks located on the diagonal and crosspeaks located at the off-diagonal position. Autopeaks areobserved when the intensities of the IR bands in the spec-trum vary as a function of the applied perturbation. Onthe other hand, synchronous cross peaks develop whentwo different IR spectral signals associated with molec-ular vibrations from different functional groups respondto the perturbation simultaneously. However, one signalmay decrease while the other is increasing. This coordi-nated response suggests strong interactions between thefunctional groups that give rise to cross peaks in the syn-chronous spectrum. In the asynchronous spectrum a crosspeak between two dynamic spectral features can developonly if their intensities vary out of phase with each other.Cross peaks usually indicate that chemical interactionsbetween the functional groups are decoupled and un-coordinated. This feature can be used to discriminatefunctional groups that exist in different local molecularenvironments, since these groups can experience differenteffects due to some external perturbations.7 In this paperwe shall focus on the amide I functional group whichshows dynamic spectral activity in the region between1600 and 1700 cm2 1.

Figure 2 shows the contour map of the synchronous(Fig. 2A) and the asynchronous (Fig. 2B) 2D IR corre-

lation spectra of b -lactoglobulin generated from all 14spectra shown in Fig. 1. The synchronous spectrum ischaracterized by four prominent autopeaks at 1616, 1650,1570, and 1717 cm2 1. Except for the apparent absence ofthe 1636-cm2 1 feature from the synchronous spectrum,all the autopeaks agree well with those identi® ed fromthe conventional absorbance spectra in Fig. 1. For con-sistency, we shall continue to refer to the peaks identi® edfrom Fig. 1, i.e., 1636 for b -sheet, 1620 for aggregatedb -strands, 1650 for a -helix, 1575 for amide II, and 1725for COOH groups. From the sign of the cross peaks it isevident that the 1650-cm2 1 band correlates positivelywith those at 1620 and 1725 cm2 1 and negatively withthat at 1575 cm2 1. The appearance of these synchronouscross peaks suggests that the solvent-induced spectral in-tensities at these frequencies are coordinated. The amide

538 Volume 51, Number 4, 1997

FIG. 3. FT-NIR absorbance spectra of b -lactoglobulin in bromoetha-nol/H2O in the combination region.

II band at 1575 cm2 1 also shows a strong positive cor-relation with the b -sheet structure at 1636 cm2 1. Noticealso that the COOH groups positively correlate with thea -helical structures. This behavior implies that, as the di-electric constant of the solvent is changed, the folding ofthe protein into a -helical structures increases the numberof exposed COOH groups.

A number of cross peaks can be identi® ed in the con-tour map of the asynchronous 2D IR correlation spectrumof b -lactoglobulin. Figure 2B shows that the band at 1575cm2 1 (amide II) is asynchronously correlated with thebands at 1620 cm2 1 ( b -aggregate) and 1650 cm2 1 ( a -he-lix), indicating that the amide II group is not associatedwith the a -helix and aggregated b -strands. Although theamide II band observed in this study is approximately 30cm2 1 greater than previously recorded bands for b -sheetstructures,10 the presence of a synchronous cross peakbetween 1575 and 1636 cm2 1 strongly suggests that theamide II band belongs to a b -sheet structure.

As pointed out earlier, the conceptual scheme devel-oped by Noda in order to induce a dynamic spectrumdoes not specify the physical nature of the effect of theperturbation on the system.7 However, the effect of eachperturbation is uniquely associated with the speci® cmechanism that relates the stimulus with the correspond-ing response of individual constituents of the system. Inthis work, the effect of varying the concentrations ofbromoethanol is to disrupt the b -structure of the protein.Since the strong H-bonding ability of bromoethanol com-petes with the polypeptide itself, bromoethanol breaks theinternal H-bonds, forcing the protein to unfold, whichexposes the CO and NH groups. These exposed groupsbecome available for the formation of aggregatedb -strands. At much higher bromoethanol concentration,the protein unfolds completely and then re-folds intoa -helix structure. These events are captured in the spectrashown in Fig. 1. A great deal of dynamic spectral activityis observed in the region between 1600 and 1700 cm2 1

as we vary the concentration of bromoethanol. As dis-cussed above, both the 1620- and 1650-cm2 1 bands ap-pear in the spectra (Fig. 1) at relatively high concentra-tion of bromoethanol followed by a continued growth ofthe 1650-cm2 1 band at the expense of both the 1636- and1620-cm2 1 bands at much higher concentrations of bro-moethanol. Since the band at around 1620 cm2 1 is attrib-uted to the formation of aggregates of b -strands, we caninfer from the spectra in Fig. 1 the following sequenceof events: b -sheets ® aggregated b -strands ® a -helix.The same behavior was observed by Jackson andMantsch in the concanavalin A/DMSO± D2O system.11

However, the results of the 2D asynchronous correlationanalysis suggest a different pattern of behavior. It mustbe pointed out that correlation cross peaks for both syn-chronous and asynchronous spectra are observed for thebands associated with the b -sheets, aggregated b -strands,and a -helix. But this consideration should not cause anyconcern, because, as long as cross peaks develop in theasynchronous spectrum, bands are deconvoluted with cer-tainty. Whereas synchronous cross peaks merely suggestpossible coordination in the responses between spectralbands, asynchronous cross peaks provide an unequivocalmessage that the responses are uncoordinated.8 Hence,the appearance of cross peaks between the bands at 1620,

1636, and 1650 cm2 1 indicates that the response patternsof the a -helix, the b -sheet, and the aggregated strands inbromoethanol solvent systems are independent of eachother. This is an unlikely situation, because the a -helixand b -aggregates can come only from the originalb -sheets. Hence any activity accompanied by changes inthe concentration of bromoethanol must be highly coor-dinated; i.e., a decrease in the intensity of the b -sheetspectral bands must be accompanied by a correspondingincrease in the intensity of the b -aggregate bands. Thesame should be true for the b -aggregates and the a -helix,since the latter results from the unfolding and re-foldingof the former. If we assume that the asynchronous crosspeaks are not simply the result of computational artifacts,then we can resolve this seemingly con¯ icting picture bypostulating that the disruption of the b secondary struc-ture (hence conversion into a -helix) occurs in terms oftwo parallel pathways, i.e., (1) b -sheets ® aggregatedb -strands ® a -helix, and (2) b -sheets ® a -helix. In thissituation, the activities of these functional groups wouldappear to be uncoordinated. Hence cross peaks woulddevelop in the asynchronous spectrum, which is observedin this study. The simultaneous appearance of the peaksat 1620 and 1650 cm2 1 (refer to the spectra in Fig. 1labeled as 0.050) seems to support this interpretation. Ithas been shown that halogenated alcohols can induce he-lical secondary structures within a polypeptide chain,12

which further supports our claim that b -sheet can be di-rectly converted into helical structures. However, we arenot prepared to demonstrate unambiguously the certaintyof this mechanism (although work is currently underwayto con® rm this mechanism).

Spectra in the NIR Region. Figure 3 shows the

APPLIED SPECTROSCOPY 539

FIG. 4. (A) Synchronous and (B) asynchronous 2D FT-NIR correlationcontour maps of b -lactoglobulin in bromoethanol/H2O. Shaded areasrepresent regions of negative correlation.

FT-NIR spectra of b -lactoglobulin in bromoethanol/H2Osolvent mixtures in the combination region. In pure H2O(MF 5 0), b -lactoglobulin possesses four characteristicabsorption bands centered at 4290, 4360, 4420, and 4610cm2 1 and a shoulder band at ; 4430 cm2 1. The ® rst threemain bands are assigned to combinations involving var-ious C± H stretching modes and C± H deformation modesof the protein side chains, while the last band at 4610cm2 1 is assigned to a combination of N± H stretching (am-ide B) and N± H bend (amide II).13,14 As the concentrationof bromoethanol is increased, a gradual increase in theintensities of the bands is observed. This increase be-comes more pronounced at high bromoethanol concen-tration. These spectral variations suggest a possible struc-tural change in the protein side groups as a result of thechanges in the dielectric constant of the solvent.

2D Correlation in the NIR Region. Figure 4A showsthe synchronous 2D NIR spectrum obtained from thespectra in Fig. 3. The spectrum reveals the presence ofthree autopeaks at 4420, 4430, and 4610 cm2 1 (notshown) and two negative cross peaks at 4300 and 4420cm2 1 and 4430 and 4610 cm2 1. Since both bands at 4300and 4420 cm2 1 correspond to combination modes of C±H stretches, a cross peak between the two simply sug-gests that the changes in the intensities of the two bandsare similar. We infer from this observation that the inter-action between the various side chains of the proteinsconstitutes an important element in the transition of theprotein from predominantly b -sheet to a -helical struc-tures. Using the same analysis, we infer from the negativecross peak at 4610 cm2 1 (amide B combined with amideII) and 4430 cm2 1 (C± H stretch and C± H deformationcombination) that reactions of the functional groups thatgive rise to these peaks towards varying concentrationsof bromoethanol are coordinated, although one is increas-ing while the other is decreasing.

The corresponding asynchronous 2D correlation spec-trum (Fig. 4B) shows a pair of cross peaks located at4420 and 4430 cm2 1 and 4430 and 4440 cm2 1. The asyn-chronous peak at 4425 extends in the region from 4340to 4440 cm2 1, indicating that the change in the C± H com-bination band in response to changing the solvent polarityis very different from the rest of the bands in that spectralregion. It is rather surprising that no asynchronous cor-relation peak is observed in the high-energy combinationregion of the spectrum. One would expect the amideB/amide II combination band to elucidate the secondarystructure of protein since protein structure is sensitive tothe hydrogen bonding between N± H and C5 O.

CONCLUSION

The results presented in this work demonstrate the po-tential of using 2D correlation spectroscopy to study themacroscopic properties of proteins in aqueous solutions.By analyzing the chemically induced spectral variationsin the amides I and II bands, we were able to detectvarious conformational structures of b -lactoglobulin inbromoethanol/D2O solution. From 2D FT-MIR correla-tion spectra, we were able to show that the mechanismfor converting b -lactoglobulin from the b -sheet to ana -helical structure involved two parallel pathways. Al-though no similar structural features were observed in theFT-NIR spectra, the results from the correlation analysisillustrated the possibility that 2D correlation analysiscould be used to investigate structural changes in pro-teins.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Gary W. Small for the use of the near-infrared spectrometer and Pete Tandler for writing the 2D correlationsubroutines.

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